JP5162574B2 - Cyclic alkylamine derivatives as inhibitors of the interaction between MDM2 and P53 - Google Patents

Abstract

The present invention provides compounds of formula (I), their use as an inhibitor of a p53-MDM2 interaction as well as pharmaceutical compositions comprising said compounds of formula (I) wherein n, m, p, t, R1, R2, R3, R4, R5, R6, R7, Q, Y and Z have defined meanings.

Description

  The present invention is directed to compounds that act as inhibitors of the interaction between MDM2 and p53 and compositions containing such compounds. Furthermore, the present invention provides: Disclosed inhibitors, methods of preparing compositions comprising them, and methods of using them, for example, as a medicament are provided.

  P53 is a tumor suppressor protein that plays a central role in controlling the balance between cell proliferation and cell growth arrest / apoptosis. Under normal conditions, the half-life of p53 is very short, so that the concentration of p53 in the cell is low. However, in response to cellular DNA damage or cellular stress (eg, oncogene activation, telomere erosion, hypoxia), p53 levels increase. This increase in p53 levels results in the activation of transcription of a number of genes, which leads the cell to either a growth arrest or apoptotic process. Thus, an important function of p53 is to inhibit the uncontrolled proliferation of damaged cells, thereby protecting the organism from cancer development.

  MDM2 is an important inhibitory regulator of p53 function. It forms a repressive autoregulatory loop by binding to the amino terminal transcriptional promotion domain of p53, thereby preventing MDM2 from p53's ability to activate transcription and targeting p53 for proteolytic degradation. Under normal conditions, this control loop contributes to maintain a low level of p53. However, in tumors with wild-type p53, the equilibrium concentration of active p53 can be increased by antagonizing the interaction between MDM2 and p53. This will result in restoration of the pro-apoptotic and anti-proliferative effects mediated by p53 in such tumor cells.

  MDM2 is a cellular proto-oncogene. Overexpression of MDM2 has been observed in various cancers. MDM2 is overexpressed in various tumors due to gene growth or increased transcription or translation. The mechanism by which MDM2 proliferation promotes tumor formation is at least in part related to its interaction with p53. In cellular overexpressed MDM2, the protective function of p53 is impeded, and therefore cells cannot respond to DNA damage or cellular stress by increasing p53 levels, leading to cell growth arrest and / or apoptosis. Thus, after DNA damage and / or cell stress, cellular overexpressed MDM2 continues to grow freely and adopt an oncogenic phenotype. Under these conditions, disruption of the interaction of p53 and MDM2 is thought to release p53 and thus function normal signals for growth arrest and / or apoptosis.

  MDM2 may also have other functions besides p53 inhibition. For example, MDM2 has been shown to interact directly with the pRb-regulated transcription factor E2F1 / DP1. This interaction is thought to be essential for the oncogenic action of MDM2 independent of p53. The domain of E2F1 shows significant homology to the MDM2 binding domain of p53. Since MDM2 interaction with both P53 and E2F1 is located at the same binding site on MDM2, MDM2 / p53 antagonists not only activate cellular p53, but also generally deregulate in tumor cells. It can be expected that it will also modulate the activity of E2F1.

Furthermore, the therapeutic effects (chemotherapy and radiation therapy) of recently used DNA damaging substances are M
It may be limited by the inhibitory control of p53 by DM2. Thus, when feedback suppression of MDM2 in p53 is hindered, an increase in functional p53 levels can therapeutically treat such agents by restoring wild-type p53 function resulting in apoptosis and / or reversal of p53-related drug resistance. Will increase the effect. Combining in vivo MDM2 inhibition and DNA damage treatment has been shown to produce a synergistic anti-tumor effect (see Non-Patent Document 1).

  Thus, disruption of the interaction of MDM2 and p53 may provide a therapeutic intervention approach in tumors containing wild-type p53, may even show an antiproliferative effect in tumor cells that do not have functional p53, and In addition, carcinogenic cells can be sensitized to chemotherapy and radiation therapy.

In particular, Patent Document 1 published on May 18, 1999 describes substituted phenylaminocarbonylindolyl derivatives as 5-HT receptor antagonists (see Patent Document 1). U.S. Patent No. 5,053,028, published on May 18, 2000, describes anthranilic acid amides useful as inhibitors of vascular endothelial growth factor receptor (VEGFR) and in the treatment of angiogenic disorders (see Patent Literature 2). . US Pat. No. 6,028,028 published on March 21, 2002 discloses tricyclic tert-amine derivatives useful as chemokine receptor CXCR4 or CCR5 modulators for processing human immunodeficiency virus and feline immunodeficiency virus. (See Patent Document 3). Patent Document 4 published on October 10, 2002 discloses N- (2-arylethyl) benzylamine as an antagonist of the 5-HT ₆ receptor (see Patent Document 4). Patent Document 5 published on March 23, 2000 provides a piperazine-4-phenyl derivative as an inhibitor of the interaction between MDM2 and p53 (see Patent Document 5). Patent Document 6 published on June 8, 2000 provides a tricyclic compound for recovering the stability of the three-dimensional structure of a p53 family protein (see Patent Document 6). Patent document 7 published on May 22, 2003 describes substituted 1,4-benzodiazepines and their use as inhibitors of MDM2-p53 interaction (see patent document 7). Patent Document 8 published on June 26, 2003 provides cis-2,4,5-triphenyl-imidazolone that inhibits the interaction of the MDM2 protein with a p53-like peptide and has an antiproliferative action (Patent Document). 8). Patent Document 9 published on January 15, 2004 discloses a bisarylsulfonamide compound that binds to MDM2 and can be used for cancer treatment (see Patent Document 9). There is a continuing need for effective and potent small molecules that inhibit the interaction between MDM2 and p53.

The compounds of the present invention differ from the prior art in structure, their pharmacological action and / or pharmacological strength.
JP 11-130750 A European Patent No. 1129074 European Patent No. 1317443 EP 1379239 International Publication No. 00/15357 Pamphlet European Patent No. 1137418 European Patent No. 1444337 European Patent No. 1458380 European Patent No. 1519932 Vousden K.M. H. , Cell, Vol. 103, 691-694, 2000

The present invention provides compounds, compositions and methods for inhibiting the interaction between MDM2 and p53 to treat cancer. Furthermore, the compounds and compositions of the present invention are useful for enhancing the effectiveness of chemotherapy and radiation therapy.

  The present invention relates to a compound of formula (I)

And their N-oxide forms, addition salts or stereochemically isomeric forms,
Where
m is 0, 1 or 2, and when m is 0, a direct bond is intended;
n is 0, 1, 2 or 3, and when n is 0, a direct bond is intended;
p is 0 or 1, and when p is 0, a direct bond is intended;
t is 0 or 1, and when t is 0, a direct bond is intended;

Is —CR ⁸ ═C <, and the dotted line is one bond, —C (═O) —CH <, —C (═O) —N <, —CHR ⁸ —CH <or CHR ⁸ —N <. Each R ⁸ is independently hydrogen or C _1-6 alkyl,
R ¹ and R ² are each independently hydrogen, halo, C _1-6 alkyl, C _1-6 alkyloxy, aryl C _1-6 alkyloxy, heteroaryl C _1-6 alkyloxy, phenylthio, hydroxy C _1-6 alkylcarbonyl; C _1-6 alkyl substituted with a substituent selected from amino, aryl and heteroaryl, or C _3-7 cyclo substituted with a substituent selected from amino, aryl and heteroaryl Selected from alkyl,
R ³ and R ⁴ are each independently selected from hydrogen, halo, C _1-6 alkyl, polyhaloC _1-6 alkyl, cyano, cyano C _1-6 alkyl, hydroxy, amino or C _1-6 alkyloxy Or R ⁴ and R ⁵ together may optionally form a divalent group selected from methylenedioxy or ethylenedioxy,
R ⁵ is hydrogen, C _1-6 alkyloxycarbonyl or C _1-6 alkyl;
R ⁶ and R ⁷ are each independently selected from hydrogen, C _1-6 alkyloxy C _1-6 alkyl or C _1-6 alkyl, or
R ⁶ and R ⁷ together may optionally
_{- (CH 2) 2 -O- (} CH 2) 2 - (b-1),
— (CH ₂ ) ₂ —NR ⁹ — (CH ₂ ) ₂ — (b-2)
May form a divalent group selected from: wherein R ⁹ is hydrogen, C _1-6 alkyloxyC _1-6 alkyl or C _1-6 alkyl,
Z is

Wherein R ¹⁰ and R ¹¹ are each independently hydrogen, halo, hydroxy, amino, C _1-6 alkyl, nitro, polyhaloC _1-6 alkyl, cyano, cyano C _{1 -6} alkyl, tetrazolo C _1-6 alkyl, aryl, heteroaryl, aryl C _1-6 alkyl, heteroaryl C _1-6 alkyl, aryl (hydroxy) C _1-6 alkyl, heteroaryl (hydroxy) C _1-6 Alkyl, arylcarbonyl, heteroarylcarbonyl, C _1-6 alkylcarbonyl, aryl C _1-6 alkylcarbonyl, heteroaryl C _1-6 alkylcarbonyl, C _1-6 alkyloxy, C _3-7 cycloalkylcarbonyl, C _{3 -7} cycloalkyl _{(hydroxy) C 1-6} alkyl, aryl _1-6 alkyloxy _{C 1-6 alkyl, C 1-6} alkyloxy _{C 1-6} alkyloxy _{C 1-6 alkyl, C 1-6} alkylcarbonyloxy _{C 1-6 alkyl, C 1-6} alkyloxycarbonyl C _1-6 alkyloxy C _1-6 alkyl, hydroxy C _1-6 alkyloxy C _1-6 alkyl, C _1-6 alkyloxycarbonyl C _2-6 alkenyl, C _1-6 alkyloxy C _1-6 alkyl, C _1-6 alkyloxycarbonyl, C _1-6 alkylcarbonyloxy, aminocarbonyl, hydroxy C _1-6 alkyl, amino C _1-6 alkyl, hydroxycarbonyl, hydroxycarbonyl C _1-6 alkyl and — (CH ₂ ) _v — ^{_{(C (= O) r)}} - is selected from _{^{(CHR 17) u -NR 13 R}} 14 Where v is 0,1,2,3,4,5 or 6, and v is intended is a direct bond when it is 0,
r is 0 or 1, and when r is 0, a direct bond is intended;
u is 0, 1, 2, 3, 4, 5 or 6, and when u is 0, a direct bond is intended;
R ¹⁷ is hydrogen or C _1-6 alkyl,
R ¹² is _{hydrogen, C 1-6 alkyl, C 3-7} cycloalkyl; hydroxy, _{amino, C 1-6} alkyloxy and _{C 1-6} alkyl substituted with a substituent selected from aryl, or a hydroxy, amino C _3-7 cycloalkyl substituted with a substituent selected from aryl, and C _1-6 alkyloxy,
R ¹³ and R ¹⁴ are each independently hydrogen, C _1-12 alkyl, C _1-6 alkylcarbonyl, C _1-6 alkylsulfonyl, aryl C _1-6 alkylcarbonyl, C _3-7 cycloalkyl, C _3-7 cycloalkylcarbonyl, — (CH ₂ ) _k —NR ¹⁵ R ¹⁶ ; a substitution selected from hydroxy, hydroxycarbonyl, cyano, C _1-6 alkyloxycarbonyl, C _1-6 alkyloxy, aryl or heteroaryl Substituted with a substituent selected from C _1-12 alkyl substituted with a group, or hydroxy, C _1-6 alkyloxy, aryl, amino, aryl C _1-6 alkyl, heteroaryl or heteroaryl C _1-6 alkyl Selected from C _3-7 cycloalkyl, or
R ¹³ and R ¹⁴ together with the nitrogen to which they are attached are optionally morpholinyl, piperidinyl, pyrrolidinyl, piperazinyl or C _1-6 alkyl, aryl C _1-6 alkyl, aryl C _1-6 Piperazinyl substituted with a substituent selected from alkyloxycarbonyl, heteroaryl C _1-6 alkyl, C _3-7 cycloalkyl and C _3-7 cycloalkyl C _1-6 alkyl may be formed, wherein k is 0, 1, 2, 3, 4, 5 or 6, and when k is 0, a direct bond is intended;
R ¹⁵ and R ¹⁶ are each independently hydrogen, C _1-6 alkyl, aryl C _1-6 alkyloxycarbonyl, C _3-7 cycloalkyl; hydroxy, C _1-6 alkyloxy, aryl and heteroaryl. With a C _1-12 alkyl substituted with a selected substituent and a substituent selected from hydroxy, C _1-6 alkyloxy, aryl, aryl C _1-6 alkyl, heteroaryl and heteroaryl C _1-6 alkyl Selected from substituted C _3-7 cycloalkyl, or
R ¹⁵ and R ¹⁶ together with the nitrogen to which they are attached may form a piperazinyl optionally substituted with morpholinyl, piperazinyl or C _1-6 alkyloxycarbonyl;
Aryl is phenyl or naphthalenyl,
Each of phenyl or naphthalenyl is optionally substituted by 1, 2 or 3 substituents each independently selected from halo, hydroxy, C _1-6 alkyl, amino, polyhalo C _1-6 alkyl and C _1-6 alkyloxy Each of the phenyl or naphthalenyl may optionally be substituted with a divalent group selected from methylenedioxy and ethylenedioxy;
Heteroaryl is pyridinyl, indolyl, quinolinyl, imidazolyl, furanyl, thienyl, oxadiazolyl, tetrazolyl, benzofuranyl or tetrahydrofuranyl;
Pyridinyl, indolyl, quinolinyl, imidazolyl, furanyl, thienyl, oxadiazolyl, tetrazolyl, benzofuranyl or tetrahydrofuranyl are each optionally halo, hydroxy, C _1-6 alkyl, amino, polyhaloC _1-6 alkyl, aryl, aryl C ₁ Optionally substituted by 1, 2 or 3 substituents each independently selected from _-6 alkyl or C _1-6 alkyloxy, and pyridinyl, indolyl, quinolinyl, imidazolyl, furanyl, thienyl, benzofuranyl or tetrahydrofuran Each nil may be optionally substituted with a divalent group selected from methylenedioxy or ethylenedioxy.

  The compounds of formula (I) can also exist in their tautomeric form. Such forms although not explicitly indicated in the above formula are intended to be included within the scope of the present invention.

Some terms used in the definitions above and thereafter are described below. These terms are sometimes used as-is or as compound terms.

In the above definitions and hereafter used halo includes fluoro, chloro, bromo and iodo and C _1-6 alkyl is for example methyl, ethyl, propyl, butyl, pentyl, hexyl, 1-methylethyl. Defined as straight and branched chain saturated hydrocarbon groups having 1 to 6 carbon atoms, such as 2-methylpropyl, 2-methyl-butyl, 2-methylpentyl and the like, and hydroxy C _1-6 alkyl Is defined as a hydroxy substituent on straight and branched chain saturated hydrocarbon groups having 1 to 6 carbon atoms, and trihalomethyl is methyl containing three identical or different halo substituents, for example is defined as trifluoromethyl, C _3-7 cycloalkyl cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl Such as cycloheptyl, etc., including cyclic hydrocarbon groups having 3 to 10 carbons.

  The term “addition salt” means that the compound of formula (I) is an organic or inorganic base such as amines, alkali metal bases and alkaline earth metal bases or quaternary ammonium bases, or mineral acids, sulfonic acids, carboxylic acids. Or a salt that can be formed with an organic or inorganic acid, such as a phosphorus-containing acid.

  The term “addition salt” further comprises pharmaceutically acceptable salts, metal complexes and solvates and salts thereof that the compounds of formula (I) can form.

  The term “pharmaceutically acceptable salt” means a pharmaceutically acceptable acid or base addition salt. The pharmaceutically acceptable acid or base addition salts referred to above comprise the therapeutically effective non-toxic acid and non-toxic base addition salt forms that the compounds of formula (I) can form. Means that. The basic compounds of formula (I) can be converted into their pharmaceutically acceptable acid addition salts by treating the base form with a suitable acid. Suitable acids are, for example, inorganic acids such as hydrohalic acids (eg hydrochloric acid or hydrobromic acid), sulfuric acid, nitric acid, phosphoric acid and the like, or eg acetic acid, propanoic acid, hydroxyacetic acid, lactic acid, pyruvic acid. , Succinic acid, malonic acid, succinic acid (ie butanedioic acid), maleic acid, fumaric acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclamic acid, An organic acid such as an acid such as salicylic acid, p-aminosalicylic acid, pamoic acid and the like. Compounds of formula (I) having acidity can be converted to their pharmaceutically acceptable base addition salts by treating the acid form with a suitable organic or inorganic base. Suitable base salt forms include, for example, ammonium salts, alkali and alkaline earth metal salts (eg, lithium, sodium, potassium, magnesium, calcium salts, etc.), salts with organic bases (eg, benzathine, N-methyl-D-glucamine, Hydrabamine salts) and salts with amino acids such as arginine, lysine and the like. The term acid or base addition salt also comprises hydrate and solvent addition forms that the compounds of formula (I) can form. Examples of such forms are, for example, hydrates, alcoholates and the like.

  The term “metal complex” means a complex formed between a compound of formula (I) and one or more organic or inorganic metal salts. Examples of the organic or inorganic salts include the second main group metal (eg, magnesium or calcium) of the periodic system, the third or fourth main group (eg, aluminum, bell, lead), and the first to eighth transitions of the periodic system. Group metals (such as chromium, manganese, iron, cobalt, nickel, copper, zinc, etc.), halides, nitrates, sulfates, phosphates, acetates, trifluoroacetates, trichloroacetates, propionates, tartrates, sulfonates For example, methyl sulfonate, 4-methylphenyl sulfonate, salicylate, benzoate and the like.

  The term “stereochemically isomeric form of a compound of formula (I)” as used above is composed of identical atoms joined by bonds of the same sequence that can be possessed by a compound of formula (I). However, it is defined as all possible compounds having different three-dimensional structures that are not interchangeable. Unless otherwise stated or stated otherwise, the chemical name of a compound includes a mixture of all possible stereochemically isomeric forms that the compound may have. The mixture may contain all diastereomers and / or enantiomers of the basic molecular structure of the compound. All stereochemically isomeric forms of both compounds of formula (I) in pure form or as mixtures with each other are intended to be included within the scope of the present invention.

  Of particular interest are stereochemically pure compounds of formula (I).

  Pure stereoisomeric forms of the compounds and intermediates described herein are defined as isomers that are substantially free of other enantiomeric or diastereomeric forms of the same basic molecular structure of the compound or intermediate. . In particular, the term “stereoisomerically pure” refers to at least 80% stereoisomer excess (ie, a minimum of 90% of one isomer and a maximum of 10% of the other possible isomers) to 100% of stereoisomers. A compound or intermediate having up to an excess (ie 100% free of one isomer and the other isomer at all), more specifically having a stereoisomeric excess of 90% to 100%, even more Specifically, it is directed to compounds or intermediates having a stereoisomer excess of 94% to 100%, and most specifically having a stereoisomer excess of 97% to 100%. The terms “enantiomerically pure” and “diastereomeric pure” are to be understood in a similar manner, but in that case understood in relation to the enantiomeric or diastereomeric excess of the mixture in question. Must.

  A tautomeric form of a compound of formula (I) is meant to comprise, for example, a compound of formula (I) in which the enol group is converted to a keto group (keto-enol tautomerism).

  N-oxide forms of the compounds of the formula (I) are those in which one or more nitrogen atoms are so-called N-oxides, in particular one or more piperidine-, piperazine or pyridazinyl-nitrogens are N-oxidised. It is meant to comprise a compound of formula (I) that has been oxidized to N-oxide.

  Compounds of formula (I) can be converted to the corresponding N-oxide form according to methods known in the art for converting trivalent nitrogen to its N-oxide form. The N-oxidation reaction can generally be carried out by reacting the starting material of formula (I) with a suitable organic or inorganic peroxide. Suitable inorganic peroxides comprise for example hydrogen peroxide, alkali metal or alkaline earth metal peroxides such as sodium peroxide, potassium peroxide, suitable organic peroxides such as benzene carboperoxo acid. Alternatively, it may comprise a halo-substituted benzene carboperoxo acid (eg 3-chlorobenzene carboperoxo acid), a peroxoalkanoic acid (eg peroxoacetic acid), an alkyl hydroperoxide (eg t-butyl hydroperoxide). Suitable solvents are, for example, lower alcohols (such as ethanol), hydrocarbons (such as toluene), ketones (such as 2-butanone), halogenated hydrocarbons (such as dichloromethane) and mixtures of such solvents.

  The present invention is also intended to include any isotopes of atoms present in the compounds of the invention. For example, hydrogen isotopes include tritium and deuterium, and carbon isotopes include C-13 and C-14.

When used hereinafter, the term “compound of formula (I)” is also meant to encompass the N-oxide form, pharmaceutically acceptable acid or base addition salts and all stereoisomeric forms. Is done.

The first group of interesting compounds consists of compounds of formula (I) to which one or more of the following constraints apply:
a) m is 0,
b) n is 2.
c) p is 1.
d) t is 0,
e)

Is -CH = C <,
f) R ¹ and R ² are each independently hydrogen.
g) R ³ and R ⁴ are each independently hydrogen,
h) R ⁵ is hydrogen,
i) R ⁶ and R ⁷ are each independently hydrogen or C _1-6 alkyl,
j) Z is a group selected from (a-1), (a-2) or (a-4), or k) R ¹⁰ and R ¹¹ are each independently hydrogen, hydroxy, C _1-6 Selected from alkyloxycarbonyl or hydroxy C _1-6 alkyl.

The second group of interesting compounds consists of compounds of formula (I) and one of the first group of interesting compounds to which one or more of the following constraints apply:
a) m is 0,
b) n is 2.
c) p is 1.
d) t is 0,
e)

Is -CH = C <,
f) R ¹ and R ² are each independently hydrogen.
g) R ³ and R ⁴ are each independently hydrogen,
h) R ⁵ is hydrogen,
i) R ⁶ and R ⁷ are each independently hydrogen,
j) Z is a group selected from (a-2) or (a-4), or k) R ¹⁰ and R ¹¹ are each independently selected from hydrogen, hydroxy or hydroxy C _1-6 alkyl. .

One group of preferred compounds is that m is 0, n is 0, p is 1, t is 0, R ¹ and R ² are each independently hydrogen, R ³ and R ⁴ is each independently hydrogen, R ⁵ is hydrogen, R ⁶ and R ⁷ are each independently hydrogen or C _1-6 alkyl, Z is (a-1), (a-2 Or a group selected from (a-4), or R ¹⁰ and R ¹¹ are each independently selected from hydrogen, hydroxy, C _1-6 alkyloxycarbonyl or hydroxy C _1-6 alkyl It consists of a compound of (I) or any subgroup thereof.

One group of more preferred compounds are those in which m is 0, n is 0, p is 1, t is 0, R ¹ and R ² are each independently hydrogen, R ³ And R ⁴ are each independently hydrogen, R ⁵ is hydrogen, R ⁶ and R ⁷ are each independently hydrogen, Z is selected from (a-2) or (a-4) It consists of compounds of the formula (I) or any subgroup thereof, which are radicals, or R ¹⁰ and R ¹¹ are each independently selected from hydrogen, hydroxy or hydroxy C _1-6 alkyl.

  The most preferred compound is compound no. 1, compound no. 4 and compound no. 5.

  The compounds of formula (I), their pharmaceutically acceptable salts and N-oxides and stereochemically isomeric forms can be prepared by conventional methods. The starting materials and some intermediates are known compounds, can be purchased commercially or can be prepared according to conventional reaction methods generally known in the art.

  Some such preparation methods will be described in more detail later. Other methods for obtaining final compounds of formula (I) are illustrated in the examples.

A compound of formula (I) is an intermediate of formula (II) wherein W is, for example, halo (eg, fluoro, chloro, bromo or iodo) or a sulfonyloxy group (methylsulfonyloxy, 4-methylphenylsulfonyloxy, etc.) By reaction with an intermediate of formula (III) which is a suitable leaving group such as The reaction is, for example, alcohol (eg, methanol, ethanol, 2-methoxy-ethanol, propanol, butanol, etc.), ether (eg, 4,4-dioxane, 1, 1′-oxybispropane, etc.), ketone (eg, 4-methyl- 2-pentanone) or a reaction inert solvent such as N, N-dimethylformamide, nitrobenzene, acetonitrile, acetic acid and the like. In order to capture the acid released during the course of the reaction, the addition of a suitable base (for example triethylamine or sodium carbonate) such as an alkali or alkaline earth metal carbonate or bicarbonate can be used. A small amount of a suitable metal iodide, such as sodium or potassium iodide, can be added to facilitate the reaction. The reaction can conveniently be carried out at a temperature between room temperature and the reflux temperature of the reaction mixture, and if desired, the reaction can be carried out under increased pressure.

  A compound of formula (I) wherein p is 1 and is referred to herein as a compound of formula (Ia) is a compound of formula (IV) with lithium aluminum hydride in a suitable solvent such as tetrahydrofuran. Can be prepared by converting the intermediate of

  The compound of formula (Ia) may also be a suitable reagent such as sodium borohydride (eg sodium tetrahydroborohydride) or polymer supported cyanotrihydroborate in a suitable solvent such as an alcohol (eg methanol). In the presence of an appropriate carboxaldehyde of formula (VI) can be prepared by reacting with an intermediate of formula (V).

  In a similar manner, a compound of formula (I) wherein t is 1 and is referred to herein as formula (Ib), can be obtained by converting an intermediate of formula (II) into an appropriate carboxaldehyde of formula (VII) It can be prepared by reacting with.

Compounds of formula (I) can also be converted into each other by reactions or functional group transformations known in the art. Some such transformations have already been described above. Other examples are hydrolysis of a carboxylic acid ester to the corresponding carboxylic acid or alcohol; hydrolysis of an amide to the corresponding carboxylic acid or amine; hydrolysis of a nitrile to the corresponding amide; imidazole or phenyl The above amino groups can be replaced with hydrogen by diazotization reactions known in the art and subsequent substitution of the diazo group with hydrogen; alcohols can be converted to esters and ethers; The amine can be converted to a secondary or tertiary amine; the double bond can be hydrogenated to the corresponding single bond; the iodo group on the phenyl group can be converted to a single group in the presence of a suitable palladium catalyst. It can be converted to an ester group by insertion of carbon oxide.

  An intermediate of formula (V), where m is 0 and is referred to herein as an intermediate of formula (Va), is a compound such as formula (VIII) in a suitable solvent such as methanol. The intermediate can be prepared by conversion with hydrazine hydrate.

  Intermediates of formula (Va) are also intermediates of formula (XVI) in the presence of a metal catalyst such as Raney nickel and a suitable reducing agent such as hydrogen in a suitable solvent such as methanol or ethanol. It can be prepared by reduction reaction of nitro to amine starting from the body.

  Intermediates of formula (X) are N ′-(ethylcarbonimidoyl) -N, N-dimethyl-1,3-propanediamine, monohydrochloric acid (EDC) and 1-hydroxy-1H-benzotriazole (HOBT). It can be prepared by reacting an intermediate of formula (XI) with an intermediate of formula (XII) in the presence of such a suitable reagent. The reaction can be carried out in a suitable solvent such as a mixture of dichloromethane and tetrahydrofuran in the presence of a base such as triethylamine.

  The intermediate of formula (VI) can be prepared by reacting the intermediate of formula (XIII) with lithium aluminum hydride in a suitable solvent such as tetrahydrofuran.

An intermediate of formula (VIII) (same as an intermediate of formula (XVI)), where t is 0 and is referred to herein as an intermediate of formula (VIII-a), is N, N-dimethylformamide In a reaction inert solvent such as in the presence of a solution of hydrogen chloride in 2-propanol, the intermediate of formula (IX) is converted to, for example, halo (eg fluoro, chloro, bromo or iodo) or C _1- It can be prepared by reacting with an intermediate of formula (XIV) which is a suitable leaving group such as ₆ alkyloxy (eg methyloxy).

Wherein R ⁶ and R ⁷ are both hydrogen and the intermediate of formula (IX), referred to herein as the intermediate of formula (IX-a), is a compound of formula (XV ) Can be prepared by converting the intermediate. The reaction can be carried out in a reaction inert solvent such as acetic acid.

  The compounds of formula (I) and some intermediates can have at least one stereogenic center in their structure. This stereogenic center can exist in the R or S configuration.

  Some compounds of formula (I) and some intermediates of the present invention may contain one asymmetric carbon atom. Pure stereochemically isomeric forms of the compounds and intermediates can be obtained by application of methods known in the art. For example, diastereomers can be separated by physical methods such as selective crystallization or chromatographic methods such as countercurrent distribution, liquid chromatography and the like. Enantiomers are first converted into a diastereomeric salt or mixture of compounds, for example by selective crystallization, supercritical liquid chromatography, or a mixture of compounds, with a suitable separating agent such as a chiral acid. The racemate is obtained by physically separating the mixture of diastereomeric salts or compounds by chromatographic methods such as liquid chromatography and finally converting the separated diastereomeric salts or compounds to the corresponding enantiomers. It can be obtained from a mixture. Pure stereochemically isomeric forms can also be obtained from the pure stereochemically isomeric forms of the appropriate intermediates and starting materials if the intervention reaction occurs stereospecifically.

  The compounds of formula (I), their pharmaceutically acceptable acid addition salts and stereoisomeric forms have valuable pharmacological properties that they inhibit the interaction between p53 and MDM2.

  The term “MDM2” is used herein to mean a protein obtained as a result of expression of the mdm2 gene. MDM2 within the meaning of this term encompasses all proteins encoded by mdm2, their variants, their alternative slice proteins and their phosphorylated proteins. Further, as used herein, “MDM2” refers to MDM2 analogues, eg, MDMX, also known as MDM4, and MDM2 homologues and analogues of other animals, eg, human homologue HDM2 or human analogue HDMX Is included.

  The term “method of inhibiting an interaction” or “inhibitor of interaction” as used herein is a method of preventing or reducing the direct or indirect binding of one or more molecules, peptides, proteins, enzymes or receptors. Or alternatively, means a method of inhibiting or reducing the normal activity of one or more molecules, peptides, proteins, enzymes or receptors.

The terms “inhibitor of p53 interaction with MDM2” or “p53-MDM2 inhibitor” are used herein as C.I. Used to describe substances that increase the expression of p53 in the article described in 1. This increase can be induced by, but not limited to, one or more of the following mechanisms of action:
-Suppress the interaction between p53 and MDM2,
-Direct binding to either MDM2 or p53 protein,
-Enzyme activity involved in interaction with upstream or downstream targets, eg kinases, or ubiquitination or SUMO modification,
-Sequestration or transport of MDM2 and p53 into different cell compartments,
A modulation of a protein that binds to MDM2, eg (but not limited to) p73, E2F-1, Rb, p21waf1 or cip1;
-Down-regulation or interference with MDM2 expression and / or MDM2 activity, eg, but not limited to its localization, post-translational modification, nuclear export or ubiquitin ligase activity,
-Direct or indirect stabilization of the p53 protein, for example by maintaining the p53 protein in its functional structural form or avoiding misfolding,
-Promoting the expression of p53 or p53 family members (eg p63 and p73),
-E.g. (but not limited to) increasing its p53 activity by promoting its transcriptional activity and / or-genes and proteins of the p53-signaling pathway, e.g. (but not limited to) p21waf1, cip1, MIC-1 ( Increased expression of GDF-15), PIG-3 and ATF-3.

  Accordingly, the present invention discloses a compound of formula (I) for use as a medicament.

  Furthermore, the present invention is also directed to the use of the compounds for the manufacture of a medicament for the treatment of disorders mediated by p53-MDM2 interaction, wherein the compound is a compound of formula (I).

  The term “treating” or “treatment” as used herein covers any treatment of a disease and / or condition in an animal, particularly a human, and (i) has a predisposition to being susceptible to the disease and / or condition. May prevent a disease and / or condition from developing in a subject that has not yet been diagnosed to have it, and (ii) suppress the disease and / or condition, ie stop its progression (Iii) methods of alleviating a disease and / or condition, ie, inducing regression of the disease and / or condition.

  The term “disorder mediated by p53-MDM2 interaction” results in inhibition of the interaction between MDM2 protein and p53 or other cellular proteins that induce apoptosis, induce cell death, or control the cell cycle Or any undesirable or harmful condition resulting from inhibition.

  The invention is also mediated by p53-MDM2 interaction by administering an effective amount of a compound of the invention to a subject in need of such treatment, such as a mammal (and more specifically a human). A method of treating a disorder that is treated.

  The compounds of the present invention can have an antiproliferative effect on tumor cells even if such cells do not contain functional p53. More specifically, the compounds of the invention can have an antiproliferative effect in tumors with wild-type p53 and / or tumors that overexpress MDM2.

  Accordingly, the present invention also provides a method of preventing tumor growth by administering an effective amount of a compound of the present invention to a subject in need of such treatment, such as a mammal (and more specifically a human). I will provide a.

Examples of tumors that can be suppressed include, but are not limited to, lung cancer (eg, adenocarcinoma, and non-small cell lung cancer), pancreatic cancer (eg, pancreatic cancer such as exocrine pancreatic cancer), colon cancer (eg, colon adenocarcinoma) And colon adenomas such as colorectal cancer), pharyngeal cancer, oral squamous cell carcinoma, tongue cancer, gastric cancer, nasopharyngeal cancer,
Hematopoietic organ tumors of the lymphatic system (eg acute lymphoblastic leukemia, B-cell lymphoma, Brukits lymphoma), non-Hodgkins lymphoma, Hodgkin's disease, myeloid leukemia (eg acute myeloid leukemia (AML)), thyroid follicular gland Cancer, myelodysplastic syndrome (MDS), tumors of mesenchymal cell source (eg fibrosarcoma and rhabdomyosarcoma), melanoma, teratocarcinoma, neuroblastoma, brain tumor, glioma, benign tumor of the skin (eg Keratinous squamous cell tumor), breast cancer (eg advanced breast cancer), kidney cancer, ovarian cancer, cervical cancer, endometrial cancer, bladder cancer, prostate cancer including advanced disease, testicular cancer, osteosarcoma, head and Includes cervical cancer and epidermal cancer.

  The compounds of the invention can also be used for the treatment and prevention of inflammatory conditions.

  Accordingly, the present invention also provides treatment of inflammatory conditions by administering an effective amount of a compound of the present invention to a subject in need of such treatment, such as a mammal (and more specifically a human). Provide methods for prevention.

  The compounds of the present invention can also be used for the treatment of autoimmune diseases and conditions. By the term “autoimmune disease” is meant any disease in which the animal's immune system reacts adversely against self-antigens. By the term “self-antigen” is meant any antibody normally found in an animal body. Representative autoimmune diseases include, but are not limited to, Hashimoto's thyroiditis, Grave's disease, multiple sclerosis, pernicious anemia, Addison's disease, insulin-dependent diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus (SLE or lupus), skin Myositis, Crohn's disease, Wegener's granuloma, antiglomerular basement membrane disease, antiphospholipid syndrome, 25 herpes zoster, allergic encephalomyelitis, glomerulonephritis, membranous glomerulonephritis, Goodpascher's syndrome, Lambert-Eaton Includes myasthenia syndrome, myasthenia gravis, bullous pemphigoid, polyendocrine disorder, Reiter's disease and Stiff-Man syndrome.

Thus, the invention also provides autoimmune diseases and conditions by administering an effective amount of a compound of the invention to a subject in need of such treatment, such as a mammal (and more specifically a human). As well as treatment of diseases associated with structurally unstable or misfolded proteins.

  The compounds of the present invention can also be useful in the treatment of diseases associated with structurally unstable or misfolded proteins. Examples of diseases associated with structurally unstable or misfolded proteins include, without limitation, cystic fibrosis (CFTR), Marfan syndrome (fibrillin), myasthenic lateral sclerosis (superoxide) Dismutase), scurvy (collagen), maple syrup urine disease (alpha-keto acid dehydrogenase complex), osteogenesis imperfecta (type 1 procollagen pro-alpha), Creutzert-Jakob disease (prion), Alheimer's disease (beta-amyloid), familial amyloidosis (lysozyme), cataract (crystallin), familial hypercholesterolemia (LDL receptor), αI-antitrypsin deficiency, Tay-Sachs disease (beta-hexose amini Dase), retinitis pigmentosa (rhodopsin) and repleconsis (insulin receptor)

  Accordingly, the present invention is also structurally unstable by administering an effective amount of a compound of the present invention to a subject in need of such treatment, such as a mammal (and more specifically a human). Methods for the treatment of diseases associated with unfolded or misfolded proteins are provided.

  In view of their useful pharmacological properties, the compounds can be formulated into various pharmaceutical dosage forms for administration purposes.

  In order to prepare the pharmaceutical compositions of the present invention, an effective amount of a particular compound in the form of a base or acid addition salt as an active ingredient can be in a wide variety of forms depending on the form of preparation desired for administration. Combined with an intimate mixture with a pharmaceutically acceptable carrier. These pharmaceutical compositions are desirably in a single dosage form suitable for administration by oral, rectal, transdermal or parenteral injection. For example, in preparing an oral dosage form composition, in the case of oral liquid preparations such as suspensions, syrups, elixirs and solutions, any such as water, glycols, oils, alcohols, etc. In the case of conventional pharmaceutical media or powders, pills, capsules and tablets, solid carriers such as starches, sugars, kaolins, lubricants, binders, disintegrants and the like can be used.

  Because of their ease of administration, tablets and capsules, in which case clearly solid pharmaceutical carriers are employed, represent the most advantageous oral dosage unit form. A carrier for parenteral compositions will usually contain at least a majority of sterile water, although it can contain other ingredients, such as to aid solubility. For example, an injection solution can be prepared in which the carrier contains physiological saline, glucose solution, or a mixture of physiological saline and glucose solution. Injectable suspensions may also be prepared, where appropriate liquid carriers, suspensions and the like can be used. In compositions suitable for transdermal administration, the carrier may optionally be combined with a small amount of any suitable additive of any nature that does not induce significant adverse effects on the skin and / or Or optionally a suitable wetting agent. The additive can facilitate administration to the skin and / or assist in preparing the desired composition. These compositions can be administered in various ways, for example as a transdermal patch, as a spot-on agent or as an ointment. It is especially advantageous to formulate the aforementioned pharmaceutical compositions in dosage unit form for ease of administration and uniformity of dosage. The dosage unit form used in the specification and claims is a pre-determined amount of each unit calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Represents a physically divided unit suitable as a single dose containing the active ingredient. Examples of such dosage unit forms are tablets (scored tablets or coated tablets), capsules, pills, powder sachets, wafers, injection solutions or suspensions, 1 teaspoon, 1 teaspoon etc. and their A plurality of separated objects.

It is especially advantageous to formulate the aforementioned pharmaceutical compositions in dosage unit form for ease of administration and uniformity of dosage. The dosage unit form used in the specification and claims contains a predetermined quantity of active ingredient calculated such that each unit produces the desired therapeutic effect in association with the required pharmaceutical carrier. Represents a physically divided unit suitable as a single dose to contain. Examples of these dosage unit forms are tablets (including knitted or coated tablets), capsules, pills, powders, wafers, injections or suspensions, 1 teaspoon, 1 teaspoon, etc. And their separated plural bodies.

  The compounds of the invention are administered in an amount sufficient to prevent interaction between MDM2 and p53 or other cellular proteins that induce apoptosis, induce cell death, or regulate the cell cycle.

  The carcinogenicity of MDM2 is determined not only by its ability to inhibit p53, but also by its ability to modulate other tumor suppressor proteins, such as the retinoblastoma protein pRb and the closely related E2F1 transcription factor. The

  Accordingly, the compounds of the invention are administered in an amount sufficient to modulate the interaction between MDM2 and the E2F transcription factor.

  One of ordinary skill in the art can readily determine the effective amount from the test results presented hereinafter. It is generally estimated that a therapeutically effective amount would be 0.005 mg / kg to 100 mg / kg body weight, and especially 0.005 mg / kg to 10 mg / kg body weight. It may be appropriate to administer the required dose in 1, 2, 3, 4 or 5 or more divided doses at appropriate intervals throughout the day. The divided doses can be formulated, for example, as unit dosage forms containing 0.5 to 500 mg, and especially 10 mg to 500 mg of active ingredient per unit dosage form.

  Another aspect of the invention contemplates a combination of a p53-MDM2 inhibitor with another anti-cancer agent, particularly for use as a medicament, more specifically in the treatment of cancer or related diseases.

For the treatment of the aforementioned conditions, the compounds of the invention can advantageously be used in combination with one or more other medicaments, more particularly other anticancer agents. Examples of anticancer agents include, but are not limited to:
A platinum coordination compound, such as cisplatin, carboplatin or oxaliplatin,
A taxane compound, such as paclitaxel or docetaxel,
A topoisomerase I inhibitor such as a camptothecin compound, such as irinotecan or topotecan,
A topoisomerase II inhibitor, such as an antitumor epipodophyllotoxin or a podophyllotoxin derivative, such as etoposide or teniposide,
An anti-tumour vinca alkaloid, such as vinblastine, vincristine or vinorelbine,
An anti-tumor nucleoside derivative, such as 5-fluorouracil, leucovorin, gemcitabine or capecitabine,
An alkylating agent such as nitrogen mustard or nitrosourea, such as cyclophosphoamide, chlorambucil, carmustine, thiotepa, mephalan or lomustine,
An anti-tumor, anthracycline derivative, such as daunorubicin, doxorubicin, doxyl, idarubicin or mitoxantrone,
A molecule that targets the IGF-1 receptor, such as picropodophyllin,
A tetracalcine derivative, such as tetrocalcin A
-Glucocorticoidene, such as prednisone,
An antibody, eg trastuzumab (HER2 antibody), ritoximab (CD20 antibody), gemtuzumab, cetoximab, pertozumab or bevacizumab,
An estrogen receptor antagonist or a selective estrogen receptor modulator, such as tamoxifen, fulvestrant, toremifene, droloxifene, faslodex or raloxyphene,
Aromatase inhibitors such as exemestane, anastrozole, letrazole and borozole,
-Differentiation agents such as retinoids, vitamin D or retinoic acid and retinoic acid metabolism inhibitors (RAMBA), for example acutane,
-DNA methyltransferase inhibitors, such as azacitidine or decitabine,-antifolates, such as premetrexed disodium,
-Antibiotics such as antinomycin D, bleomycin, mitomycin C, dactinomycin, carminomycin or daunomycin,
-Antimetabolites such as clofarabine, aminopterin, cytosine arabinoside or methotrexate,
Anti-angiogenic agents such as apoptosis-inducing agents and Bcl-2 inhibitors, such as YC137, BH312, ABT737, Gossypol, HA 14-1, TW 37 or decanoic acid,
A tubulin binding agent such as combrestatin, colxin or nocodazole,
-Kinase inhibitors, such as flavoperidol, imatinib mesylate, erlotinib or gefitinib,
-Farnesyltransferase inhibitors, such as tipifarnib,
A histone deacetylase (HDAC) inhibitor, such as sodium butyrate, suberoylanilide hydroxamic acid (SAHA), depsipeptide (FR901228), NVP-LAQ824, R306465, JNJ-26481585 or trichostatin A,
An inhibitor of the ubiquitin-proteasome pathway, such as PS-341, MLN 41 or bortezomib,
-Yondelis,
A telomerase inhibitor, such as telomestatin,
A matrix metalloproteinase inhibitor, such as bachimasto, marimastat, purinostart or metastart,
It is.

  As noted above, the compounds of the present invention also have therapeutic applications in sensitizing tumor cells for chemotherapy and radiation therapy.

  Accordingly, the compounds of the present invention can be used as “radiosensitizers” and / or “chemosensitizers” or alternatively as “radiosensitizers” and / or “chemosensitizers”. It can be administered in combination with a “sensitizing agent”.

  The term “radiosensitizer” as used herein is administered to an animal in a therapeutically effective amount that increases the sensitivity of cells to ionizing radiation and / or facilitates treatment of diseases treatable with ionizing radiation. Defined as a molecule, preferably a low molecular weight molecule.

  The term “chemosensitizer” as used herein is administered to an animal in a therapeutically effective amount that increases the sensitivity of the cells to compound therapy and / or facilitates treatment of a disease treatable with chemotherapy. Molecule, preferably a low molecular weight molecule.

Hypoxic cell radiosensitizers that mimic oxygen or behave like bioreductants under hypoxia (eg, 2-nitroimidazole compounds and benzotriazine dioxide compounds); non-hypoxic cell radiosensitizers ( For example, halogenated pyrimidines) can be analogs of DNA bases that are preferentially incorporated into the DNA of cancer cells, thereby facilitating radiation-induced destruction of DNA molecules and / or normal DNA repair. Several mechanisms for the action of radiosensitizers have been suggested in the literature, including the ability to interfere with the mechanism; and various other possible mechanisms of action have been proposed for radiosensitization in the treatment of disease. It has been assumed for sensitizers. A number of cancer treatment protocols have recently used radiosensitizers along with x-ray irradiation. Examples of x-ray activated radiosensitizers include, but are not limited to: metronidazole, misonidazole, desmethylmisonazole, pimonidazole, etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233, EO9, RB 6145, nicotinamide, 5-bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IUdR), bromodeoxycytidine, fluorodeoxyuridine (FudR), hydroxyurea, cisplatin and their therapeutically effective analogs and Includes derivatives. Photodynamic therapy (PDT) for cancer uses visible light as a radiation activator for sensitizers. Examples of photodynamic radiosensitizers include, but are not limited to: hematoporphyrin derivatives, photofrin, benzoporphyrin derivatives, tin etioporphyrin, pheophorbide-a, bacteriochlorophyll-a, naphthalocyanine, phthalocyanine, zinc phthalocyanine and These therapeutically effective analogs and derivatives are included. Radiosensitizers include, but are not limited to, compounds that promote uptake of radiosensitizers into target cells, therapeutic agents to target cells, compounds that control nutrient and / or oxygen flow, further irradiation A therapeutically effective amount of one or more, including a chemotherapeutic agent that acts against the tumor with or without, or other therapeutically effective compounds for treating cancer or other diseases It can be administered together with other compounds.

  Chemosensitizers include, but are not limited to, compounds that promote uptake of chemotherapy sensitizers into target cells, therapeutic agents to target cells, compounds that regulate nutrient and / or oxygen flow, tumors Administration with a therapeutically effective amount of one or more other compounds, including chemotherapeutic agents acting on or other therapeutically effective compounds for treating cancer or other diseases can do. Calcium antagonists such as verapamil are useful in combination with anticancer drugs to establish chemosensitivity in tumor cells resistant to approved chemotherapeutic agents and to enhance the effects of such compounds in drug-sensitive malignancies Is found to be.

  In view of their useful pharmacological properties, the components of the combination according to the invention, ie other medicaments and p53-MDM inhibitors, can be formulated into various dosage forms for administration purposes. The components can be formulated separately into individual pharmaceutical compositions or into a single pharmaceutical composition containing both components.

  Accordingly, the present invention is also directed to a pharmaceutical composition comprising another medicament and a p53-MDM inhibitor together with one or more pharmaceutical carriers.

  The invention is further directed to the use of the combination according to the invention in the manufacture of a pharmaceutical composition for preventing the growth of tumor cells.

  The present invention further provides a p53-MDM2 inhibitor according to the present invention as a first active ingredient and a second as a combined preparation for simultaneous, separate or sequential use in the treatment of patients suffering from cancer Intended for products containing anticancer agents as active ingredients.

The other medicament and the p53-MDM2 inhibitor can be administered simultaneously (eg, in separate or single compositions) or sequentially in any order. In the latter case, the two compounds will be administered in a sufficient amount and manner within a period sufficient to ensure that an advantageous or synergistic effect is achieved. The preferred method and order of administration for each component of the combination, and the respective dosage and schedule, will depend on the particular other drug being administered and the p53-MDM2 inhibitor, their route of administration, the particular tumor being treated and the treatment. Will depend on the particular host being used. The optimal method and sequence of administration, as well as the dosage and schedule, can be readily determined by one skilled in the art using conventional methods and in view of the information presented herein.

The platinum coordination compound is advantageously 1-500 mg (mg / m ² ) per square meter of body surface area per course of treatment, for example 50-400 mg / m ² , especially about 75 mg / m ² for cisplatin. And for carboplatin at a dose of about 300 mg / m ² .

Taxane compound is advantageously, one course per treatment, 50 to 400 mg per body surface area meter (mg ^{/ m} 2), for example 75 to 250 mg / ^{m 2,} particularly for paclitaxel approximately 175~250mg / ^{m 2} , and for docetaxel is administered at a dose of about 75 to 150 mg / ^{m 2.}

The camptothecin compound is advantageously 0.1 to 400 mg (mg / m ² ) per square meter of body surface area per course of treatment, for example 1 to 300 mg / m ² , especially about 100 to 350 mg / m ² for irinotecan. m ² and for topotecan administered at a dose of about 1-2 mg / m ² .

The anti-tumor podophyllotoxin derivative is advantageously 30-300 mg (mg / m ² ) per square meter of body surface area per course of treatment, eg 50-250 mg / m ² , especially about 35 for etoposide. Administered at a dose of ˜100 mg / m ² and for teniposide about 50-250 mg / m ² .

Advantageously the anti-tumor vinca alkaloids, per treatment one course, 2～30Mg per body surface area meter (mg ^{/ m} 2), particularly for vinblastine in a dosage of about 3 to 12 mg / ^{m 2,} relative to vincristine about 1-2 mg / ^{m 2,} and for vinorelbine is administered at a dose of about 10 to 30 mg / ^{m 2.}

The preferred anti-tumor nucleoside derivative is one course per treatment, 200～2500Mg per body surface area meter (mg ^{/ m} 2), about 200 for example 700 to 1500 / ^{m 2,} particularly for 5-FU 500 mg / ^{m 2,} for the gemcitabine about 800 to 1200 mg / ^{m 2,} and for capecitabine is administered at a dose of about 1000 to 2500 / ^{m 2.}

Alkylating agents such as nitrogen mustard or nitrosourea are advantageously 100-500 mg (mg / m ² ) per square meter of body surface area per course of treatment, for example 120-200 mg / m ² , especially cyclophospho About 100-500 mg / m ² for amide, about 0.1-0.2 mg / kg for chlorambucil, about 150-200 mg / m ² for carmustine, and about 100 for lomustine. It is administered at a dose of ˜150 mg / m ² .

The anti-tumor anthracycline derivative is advantageously 10-75 mg (mg / m ² ) per square meter of body surface area per course of treatment, for example 15-60 mg / m ² , especially about 40-75 mg for doxorubicin. / ^{m 2,} about 25~45mg / ^{m 2} for the daunorubicin, and for the idarubicin is administered at a dose of about 10 to 15 mg / ^{m 2.}

  Antiestrogens are advantageously administered at a dose of about 1-100 mg per day, depending on the particular drug and the condition being treated. Tamoxifen is advantageously administered orally in a dose of 5-50 mg, preferably 10-20 mg, twice a day, continuing the treatment for a period sufficient to achieve and maintain a therapeutic effect. Toremifene is advantageously administered orally in a dosage of about 60 mg once a day and continues treatment for a period sufficient to achieve and maintain a therapeutic effect. Anastrozole is advantageously administered orally in a dosage of about 1 mg once a day. Dororoxyphene is advantageously administered orally in a dosage of about 20-100 mg once a day. Raloxyphene is advantageously administered orally in a dosage of about 60 mg once a day. Exemestane is advantageously administered orally in a dose of about 25 mg once a day.

The antibody is advantageously administered at a dosage of about 1-5 mg (mg / m ² ) per square meter of body surface area per course of treatment or as known in the art if different. Trastuzumab is advantageously per treatment of 1 course, 1 to 5 mg per body surface area meter (mg ^{/ m} 2), in particular is administered at a dose of 2-4 mg / ^{m 2.}

  These doses can be administered for example once, twice or more per course of treatment, which can be repeated for example every 7, 14, 21 or 28 days.

  The compounds of formula (I), their pharmaceutically acceptable acid addition salts and stereoisomeric forms, are labeled compounds and / or p53 and / or MDM2 and / or other molecules, peptides, proteins, Having valuable diagnostic properties that can be used to detect or identify p53-MDM2 interactions in biological samples, comprising methods for detecting or measuring the formation of complexes between enzymes or receptors be able to.

The detection or identification method can use a compound labeled with a labeling agent such as a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, and the like. Examples of radioisotopes include ¹²⁵ I, ¹³¹ I, ³ H and ¹⁴ C. Enzymes are usually made detectable by conjugation of a suitable substrate that in turn catalyzes a detectable reaction. Examples include, for example, beta-galactosidase, beta-glucosidase, alkaline phosphatase, peroxidase and malate dehydrogenase, preferably horseradish peroxidase. Luminescent substances include, for example, luminol, luminol derivatives, luciferin, ecroline and luciferase. A biological sample can be defined as body tissue or fluid. Examples of body fluids are cerebrospinal fluid, blood, plasma, serum, urine, sputum, saliva and the like.

  The following examples illustrate the present invention.

After the experimental department , “DMF” is defined as N, N-dimethylformamide, “DCM” as dichloromethane, “EtOAc” as ethyl acetate, “EtOH” as ethanol, “MeOH” as methanol, and “THF” as tetrahydrofuran.

Melting points The melting points for some compounds were obtained with a Kofler hot bench consisting of a heating plate with a linear temperature gradient, a sliding indicator and a temperature scale in Celsius.

LCMS
Method A
The HPLC gradient was measured using four pumps with a deaerator (a quarterary pump).
), Supplied by an Alliance HT 2795 (Waters) system comprising an automatic sample collector and a diode-array detector (DAD). The stream from the column was separated into the MS detector. The MS detector incorporated an electron spray ionization source. The capillary needle voltage was 3 kV and the power supply temperature was maintained at 100 ° C. on LCT (Flight-Z-spray mass spectrometer time from Waters). Nitrogen was used as the nebulizer gas. Data acquisition was performed using a Waters-Micromass MassLynx-Openlynx data system. Reverse running HPLC was performed on an Xterra-RP C18 column (5 μm, 3.9 × 150 mm) at a temperature of 30 ° C. and a flow rate of 1.0 ml / min. Using two mobile phases (mobile phase A: 100% 7 mM ammonium acetate, mobile phase B: 100% acetonitrile), 85% A, 15% B (maintained for 3 minutes) to 20% A, 80% B It was run on a gradient of (5 minutes), maintained at 20% A and 80% B for 6 minutes, and re-equilibrated for 3 minutes at the initial conditions. An injection volume of 20 μl was used. The cone voltage was 20 V for positive ionization mode. Mass spectra were acquired by scanning from 100 to 900 in 0.8 seconds using an interscan delay of 0.08 seconds.

Method B
The LC gradient was supplied by an Acquity UPLC (Waters) system comprising a dual pump, sample organizer, column heater (set at 55 ° C.) and a diode-array detector (DAD). The stream from the column was separated into the MS detector. The MS detector incorporated an electron spray ionizing light source. Mass spectra were acquired by scanning 100-1000 in 0.18 seconds using a dwell time of 0.02 seconds. The capillary needle voltage was 3.5 kV and the power supply temperature was maintained at 140 ° C. Nitrogen was used as the nebulizer gas. Data acquisition was performed using a Waters-Micromass MassLynx-Openlynx data system. Reverse run UPLC was performed on a crosslinked ethylsiloxane / silica (BEH) C18 column (1.7 μm, 2.1 × 50 mm) at a flow rate of 0.8 ml / min. Using two mobile phases (mobile phase A: 0.1% formic acid (H ₂ O / methanol in 95/5), mobile phase B: methanol), 95% A to 5% A, 95% The vehicle was run with a slope of B (1.3 minutes) and maintained for 0.2 minutes. An injection volume of 0.5 μl was used. The cone voltage was 10V for the positive ionization mode and 20V for the negative ionization mode.

A. Preparation of intermediate compound Example A1
a) Preparation of Intermediate 1

To a solution of 5-nitroindoline (10.0 g, 0.061 mol) and 4-chloropyridine hydrochloride (11.0 g, 0.073 mol) in 60 ml of DMF at 0 ° C. under argon, potassium tert- Butoxide (17.0 g, 0.15 mol) was added dropwise. The mixture was heated at 100 ° C. for 16 hours. The mixture was poured into ice and extracted twice with EtOAc. The organic layer was separated, dried (MgSO ₄ ), filtered and the solvent was evaporated. The residue was purified twice by column chromatography over silica gel (40-63 μm) (eluent: cyclohexane / EtOAc / MeOH 50/50/0 to 0/80/20). The pure fractions were collected and the solvent was evaporated, yielding 2.49 g (17%) of intermediate 1 as a brown-orange solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 8.47 (dd, 2H, J = 6.4,
J = 1.6), 8.06 (m, 2H), 7.41 (d, 1H, J = 9.6), 7.28 (dd, 2H, J = 6.4, J = 1.6) ), 4.16 (t, 2H, J = 8.6), 3.22 (t, 2H, J = 8.6).
MS (ES +) m / z 242 (M + 1).
b) Preparation of intermediate 2

A mixture of Intermediate 1 (2.4 g, 0.010 mol) and Raney nickel (5 ml, 50% slurry in water) (in 45 ml EtOH and 45 ml THF) was stirred at room temperature under 30 psi hydrogen for 3 hours. After filtration through a celite pad, the solvent was evaporated, yielding 2.01 g (96%) of intermediate 2 as a brown solid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 8.32 (dd, 2H, J = 6.4, J = 1.5), 7.15 (d, 1H, J = 8.5), 6.92 (dd , 2H, J = 6.4, J = 1.5), 6.63 (d, 1H, J = 2.2), 6.50 (dd, 1H, J = 8.3, J = 2.4). ), 3.92 (t, 2H, J = 8.3), 3.44 (brs, 2H), 3.08 (t, 2H, J = 8.3).
c) Preparation of intermediate 3

To a solution of intermediate 2 (1.9 g, 0.0089 mol) under argon (in 20 ml DCM and 20 ml THF) was added indole-3-acetic acid (2.0 g, 0.012 mol), 1-hydroxybenzo. Triazole hydrate (1.6 g, 0.012 mol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (2.2 g, 0.012 mol) were added sequentially. The reaction mixture was stirred at room temperature for 40 hours. To complete the reaction, indole-3-acetic acid (1.6 g, 0.0089 mol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (1.7 g, 0.0089 mmol) were added. And the mixture was stirred at room temperature for 16 hours. The solvent was evaporated and the residue was purified by column chromatography over silica gel (40-63 μm) (eluent: DCM / MeOH / NH ₄ OH 95/5 / 0.1-80 / 20 / 0.1). The collected fractions were evaporated and the resulting solid was washed with MeOH and dried, yielding 1.95 g (60%) of intermediate 3.
¹ H NMR (300 MHz, DMSO-d ₆ ) δ 10.93 (brs, 1H), 10.08 (brs, 1H), 8.31 (d, 2H, J = 6.3), 7.88 (d, 1H, J = 8.1), 7.58 (m, 2H), 7.34 (m, 2H), 7.26 (d, 1H, J = 2.1), 7.17 (d, 2H, J = 6.6), 7.07 (t, 1H, J = 6.9), 6.98 (t, 1H, J = 7.4), 3.99 (t, 2H, J = 8.3) ), 3.71 (s, 2H), 3.12 (t, 2H, J = 8.2).
MS (ES +) m / z 369 (M + 1).

Example A2
a) Preparation of Intermediate 4

To a solution of 5-nitroindoline (5.0 g, 0.030 mol) and 4-chloroquinoline (6.0 g, 0.037 mol) under argon (in 30 ml DMF) was added potassium tert-butoxide (8.4 g). 0.075 mol) was added dropwise. The mixture was stirred at 100 ° C. for 16 hours and then at room temperature for 70 hours. The mixture was poured into ice and extracted 3 times with EtOAc. The organic layer was separated, washed with saline, dried (MgSO ₄ ), filtered and the solvent was evaporated. The residue was purified by column chromatography over silica gel (40-63 μm) (eluent: EtOAc / cyclohexane 50 / 50-100 / 0). The pure fractions were collected and the solvent was evaporated, yielding 2.26 g (26%) of intermediate 4 as an orange solid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 8.93 (d, 1H, J = 4.9), 8.11 (m, 2H), 7.92 (dd, 1H, J = 8.9, J = 2) .5), 7.85 (d, 1H, J = 7.7), 7.82 (dt, 1H, J = 7.0, J = 1.4), 7.60 (dt, 1H, J = 6.8, J = 1.2), 7.54 (d, 1H, J = 4.9), 6.26 (d, 1H, J = 8.9), 4.30 (t, 2H, J = 8.4), 3.35 (t, 2H, J = 8.2).
MS (ES +) m / z 292 (M + 1).
b) Preparation of intermediate 5

A mixture of Intermediate 4 (2.0 g, 0.0069 mol) and Raney nickel (3 ml, 50% slurry in water) (in 30 ml EtOH and 30 ml THF) was stirred at room temperature under 30 psi hydrogen for 4.5 hours. After filtration through a celite pad, the solvent was evaporated, yielding 1.81 g (100%) of intermediate 5 as an orange solid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 8.71 (d, 1H, J = 5.1), 8.07 (d, 1H, J = 8.5), 8.01 (d, 1H, J = 8) .5), 7.67 (t, 1H, J = 7.0), 7.41 (t, 1H, J = 7.1), 7.09 (d, 1H, J = 5.1), 6 .67 (s, 1H), 6.49 (d, 1H, J = 8.3), 6.38 (dd, 1H, J = 8.3, J = 1.9), 4.04 (t, 2H, J = 7.9), 3.39 (brs, 2H), 3.12 (t, 2H, J = 7.8).
MS (ES +) m / z 262 (M + 1).
c) Preparation of intermediate 6

To a solution of intermediate 5 (1.7 g, 0.0064 mol) in argon (in 15 ml DCM and 15 ml THF) was added indole-3-acetic acid (1.5 g, 0.0084 mol), 1-hydroxybenzo. Triazole hydrate (1.1 g, 0.0084 mol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloric acid (1.6 g, 0.0084 mol) were added sequentially. The mixture was stirred at room temperature for 16 hours. The solvent was evaporated and the residue was purified by column chromatography over silica gel (40-63 μm) (eluent: DCM / MeOH / NH ₄ OH 90/10 / 0.1). The pure fractions were collected and the solvent was evaporated, yielding 1.15 g (43%) of intermediate 6 as a brown foam.
¹ H NMR (300 MHz, CDCl ₃ ) δ 8.74 (d, 1H, J = 5.1), 8.61 (brs, 1H), 8.09 (d, 1H, J = 8.1), 7. 91 (d, 1H, J = 8.4), 7.70-7.62 (m, 2H), 7.41 (m, 4H), 7.22 (m, 3H), 7.09 (d, 1H, J = 5.1), 6.74 (dd, 1H, J = 8.4, J = 1.8), 6.38 (d, 1H, J = 8.4), 4.04 (t , 2H, J = 8.0), 3.89 (s, 2H), 3.15 (t, 2H, J = 7.9).
MS (ES +) m / z 419 (M + 1).
Example A3
a) Preparation of Intermediate 7

A mixture of indole-3-acetic acid (2.0 g, 0.013 mol) and 1,1-carbonyldiimidazole (2.1 g, 0.013 mol, portionwise addition) in 28 ml of DCM at room temperature under argon Stir for 2 hours. N, O-dimethylhydroxylamine hydrochloride (1.3 g, 0.013 mol) was added and the mixture was stirred at room temperature for 16 hours and then poured into ice and water. The pH was adjusted to 10 with 4N sodium hydroxide solution and the aqueous layer was extracted twice with EtOAc. The organic layer was separated, washed with 3N hydrochloric acid solution, dried (MgSO ₄ ), filtered and the solvent was evaporated, yielding 2.37 g (89%) of intermediate 7 as a pink solid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 8.09 (brs, 1H), 7.66 (d, 1H, J = 7.5), 7.36 (d, 1H, J = 7.5), 7. 22-7.10 (m, 3H), 3.92 (s, 2H), 3.66 (s, 3H), 3.23 (s, 3H).
Example A4
a) Preparation of Intermediate 8

A mixture of 5-nitroindoline (4.0 g, 0.024 mol) and 4-chloro-2-pyridinecarboxylic acid methyl ester (5.0 g, 0.029 mol) in 24 ml acetic acid was added to the Biotage Initiator microwave apparatus. Heated to 120 ° C. for 25 minutes. The reaction was quenched with ice and the pH adjusted to pH 9 by the addition of a saturated solution of potassium carbonate. The mixture was extracted 3 times with DCM. The organic layer was separated, dried (MgSO ₄ ), filtered and the solvent was evaporated. The residue was purified by column chromatography over silica gel (40-63 μm) (eluent: DCM / MeOH 100/0 to 90/10). The pure fractions were collected and the solvent was evaporated, yielding 1.37 g (19%) of intermediate 8 as a yellow solid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 8.65 (d, 1H, J = 5.6), 8.16 (dd, 1H, J = 8.9, J = 2.1), 8.11 (s , 1H), 7.98 (d, 1H, J = 2.5), 7.35 (d, 1H, J = 8.9), 7.30 (dd, 1H, J = 5.7, J = 2.5), 4.23 (t, 2H, J = 8.5), 4.05 (s, 3H), 3.32 (t, 2H, J = 8.4).
MS (ES +) m / z 300 (M + 1).
b) Preparation of intermediate 9

A mixture of Intermediate 8 (1.2 g, 0.0045 mol) and Raney nickel (4 ml, 50% slurry in water) (in 20 ml MeOH and 20 ml THF) was stirred at room temperature under 1 atmosphere of hydrogen for 20 hours. After filtration through a celite pad, the solvent was evaporated, yielding 1.06 g (88%) of intermediate 9 as an orange solid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 8.40 (d, 1H, J = 5.8), 7.77 (d, 1H, J = 2.5), 7.18 (d, 1H, J = 8) .4), 7.05 (dd, 1H, J = 5.8, J = 2.6), 6.62 (d, 1H, J = 2.2), 6.52 (dd, 1H, J = 8.4, J = 2.4), 4.00 (m, 5H), 3.09 (t, 2H, J = 8.3).
MS (ES +) m / z 270 (M + 1).
Example A5
a) Preparation of intermediate 10

A mixture of 5-aminoindole (1.1 g, 0.0086 mol) and phthalic anhydride (2.6 g, 0.017 mol) in 20 ml DMF was stirred at 100 ° C. for 5 hours and then at room temperature for 64 hours. did. The reaction mixture was diluted in EtOAc, washed twice with a saturated solution of ammonium chloride, dried (MgSO ₄ ), filtered and the solvent was evaporated. The resulting oil was taken up in acetic acid (15 ml), stirred at room temperature for 20 minutes and then heated to 80 ° C. for 1.5 hours. Ice was added and the pH was adjusted to 4 with a saturated solution of sodium carbonate. The mixture was extracted twice with EtOAc. The organic layer was separated, dried (MgSO ₄ ), filtered and the solvent was evaporated. The residue was purified by column chromatography over silica gel (40-63 μm) (eluent: EtOAc / cyclohexane / 40/60). The pure fractions were collected and the solvent was evaporated, yielding 2.06 g (91%) of intermediate 10.
¹ H NMR (300 MHz, CDCl ₃ ) δ 8.32 (brs, 1H), 7.97 (m, 2H), 7.79 (m, 2H), 7.66 (d, 1H, J = 2.0) 7.49 (d, 1H, J = 8.6), 7.27 (dd, 1H, J = 5.7, J = 2.8), 7.17 (dd, 1H, J = 8.6) , J = 2.0), 6.61 (t, 1H, J = 1.1).
MS (ES +) m / z 263 (M + 1).
b) Preparation of intermediate 11

A mixture of intermediate 10 (2.1 g, 0.0079 mol) and sodium cyanoborohydride (987 mg, 0.016 mol) (in 30 ml acetic acid) was stirred at room temperature for 18 hours. Ice was added and the pH was adjusted to 6 with a saturated solution of sodium carbonate. The mixture was extracted twice with EtOAc. The organic layer was separated, washed with saline, dried (MgSO ₄ ), filtered and the solvent was evaporated. The residue was purified by column chromatography over silica gel (40-63 μm) (eluent: EtOAc / cyclohexane / 30 / 70-70 / 30). The pure fractions were collected and the solvent was evaporated, yielding 726 mg (35%) of intermediate 11 as a yellow solid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 7.93 (m, 2H), 7.77 (m, 2H), 7.09 (s, 1H), 7.00 (d, 1H, J = 8.2) 6.70 (d, 1H, J = 8.2), 3.62 (t, 2H, J = 8.4), 3.09 (t, 2H, J = 8.4).
MS (ES +) m / z 265 (M + 1).
c) Preparation of intermediate 12

Intermediate 11 (570 mg, 0.0022 mol), 4-bromo-6,7-dihydro-5H- [1] pyridin-7-ol (554 mg, 0.0026 mol) and 5N hydrochloric acid solution in 2-propanol A mixture (0.57 ml, 0.0029 mol) in 11 ml DMF was heated to 120 ° C. for 1 hour in a Biotage Initiator microwave apparatus. The reaction was quenched with a saturated solution of sodium bicarbonate and extracted three times with DCM. The solvent was evaporated. The residue was purified by column chromatography over silica gel (40-63 μm) (eluent: EtOAc / MeOH 100/0 to 80/20, then DCM / MeOH 95/5 to 90/10). The pure fractions were collected and the solvent was evaporated, yielding 651 mg (76%) of intermediate 12 as a yellow solid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 8.30 (d, 1H, J = 5.6), 7.95 (m, 2H), 7.79 (m, 2H), 7.23-7.12 ( m, 3H), 6.94 (d, 1H, J = 8.4), 5.24 (t, 1H, J = 7.0), 4.18 (m, 1H), 4.08 (m, 1H), 3.25 (m, 2H), 3.00 (m, 1H), 2.85 (m, 1H), 2.57 (m, 1H), 2.06 (m, 1H).
MS (ES +) m / z 398 (M + 1).
d) Preparation of intermediate 13

Room temperature hydrazine hydrate (137 μl, 0.0028 mol) was added to a suspension of intermediate 12 (557 mg, 0.0014 mol) in 5.5 ml MeOH and the resulting mixture was brought to 70 ° C. Heated for 40 minutes. The reaction was quenched with water and extracted 4 times with EtOAc. The organic layer was separated, dried (MgSO ₄ ), filtered and the solvent was evaporated, yielding 392 mg (100%) of intermediate 13 as a brown-orange solid.
¹ H NMR (300 MHz, CDCl ₃ ) δ 8.18 (d, 1H, J = 5.8), 7.07 (d, 1H, J = 5.8), 6.49 (d, 1H, J = 8) .3), 6.63 (d, 1H, J = 2.1), 6.48 (dd, 1H, J = 8.3, J = 2.4), 5.21 (m, 1H), 4 .19 (brs, 2H), 4.03 (m, 3H), 3.05 (m, 3H), 2.84 (m, 1H), 2.50 (m, 1H), 2.03 (m, 1H).
Example A6
a) Preparation of intermediate 14

Mixture of 2,3-dihydro-3,3-dimethyl-5-nitro-1H-indole (0.003 mol) and 4-bromo-pyridine (0.003 mol) in 5 ml of 1-butanol in the microwave Heat in an oven in a sealed tube to 140 ° C. for 30 minutes, then take up in 10% potassium carbonate solution and extract with EtOAc. The organic layer was washed with water, then NaCl and saline, dried (MgSO ₄ ), filtered and the solvent was evaporated. The residue (0.8 g) was purified by column chromatography over silica gel (20-45 μm) (eluent: DCM / MeOH 100/0 to 98/2). The pure fractions were collected and the solvent was evaporated. The residue (0.45g, 64%) was crystallized from acetonitrile. The precipitate was filtered off and dried, yielding 0.216g (31%) of intermediate 14, melting point 217 ° C (Kofler).
¹ H NMR (DMSO-d6) δ1.4 (6H, s), 3.92 (2H, s), 7.3 (2H, m), 7.45 (1H, m), 8.10 (2H, m), 8.47 (2H, m)
LCMS (ES +) m / z 270 (M + 1), R _t = 0.77, Method B
b) Preparation of intermediate 15

A mixture of intermediate 14 (0.003 mol) and Raney nickel (0.9 g) (in 20 ml MeOH) was hydrogenated at 3 bar pressure for 1 hour at room temperature and then filtered over celite. The celite was washed with MeOH. The filtrate was evaporated, yielding 0.8 g (100%) of intermediate 15.
c) Preparation of intermediate 16

Bromotris (pyrrolidino) phosphonium hexafluorophosphate (0.004 mol) was added to intermediate 15 (0.003 mol), 1H-indole-3-acetic acid (0.004 mol), 1-hydroxybenzotriazole (0.04 mol) ) And diisopropyl ether (0.005 mol) in 15 ml DCM was added portionwise at room temperature. The mixture was stirred overnight at room temperature. The organic layer is washed with 10% potassium carbonate solution, dried (MgSO ₄ ),
Filter and evaporate the solvent. The residue (3.2 g) on silica gel (15-40 [mu] m) on column chromatography _{(eluent: DCM / MeOH / NH 4 OH} 95/5 / 0.2) was purified by. The pure fractions were collected and the solvent was evaporated. The residue (0.7g, 52%) was crystallized from acetonitrile. The precipitate was filtered off and dried, yielding 0.55 g (41%) of intermediate 16, melting point 158 ° C. (Kofler).
¹ H NMR (DMSO-d6) δ 1.28 (6H, s), 3.7 (2H, s), 3.73 (2H, s), 6.99 (1H, t, J = 7.7 Hz), 7,07-7.11 (3H, m), 2.25 (1H, d, J = 3.6 Hz), 7.30-7.39 (3H, m), 7.55 (1H, br, d , J = 3.6 Hz), 7.62 (1H, d, J = 7.7 Hz), 8.3 (2H, d, J = 7.7 Hz), 10.03 (1H, br, s), 10 .92 (1H, br, s)
LCMS (ES +) m / z 397 (M + 1), R _t = 8.43, Method A
B. Preparation of final compound Example B1 Preparation of compound 1

Lithium aluminum hydride (423 mg, 0.0011 mol) was added portionwise under argon to a suspension of Intermediate 3 (1.0 g, 0.0027 mol) in 60 ml THF. The mixture was stirred at room temperature for 24 hours, quenched with ice and dilute Rochelle salt solution and extracted three times with DCM. The organic layer was separated, dried (MgSO ₄ ), filtered and the solvent was evaporated. The residue was purified by column chromatography over silica gel (40-63 μm) (eluent: DCM / MeOH / NH ₄ OH 95/5 / 0.1). The pure fractions were collected and the solvent was evaporated, yielding 170 mg (18%) of compound 1 as a yellow foam.
¹ H NMR (300 MHz, CDCl ₃ ) δ 8.36 (brs, 1H), 8.32 (d, 2H, J = 6.6), 7.63 (d, 1H, J = 7.8), 7. 39 (d, 1H, J = 7.5), 7.24-7.11 (m, 3H), 7.07 (d, 1H, J = 1.8), 6.93 (d, 2H, J = 6.6), 6.58 (s, 1H), 6.45 (dd, 1H, J = 8.4, J = 2.1), 3.93 (t, 2H, J = 8.2) 3.62 (brs, 1H), 3.46 (t, 2H, J = 7.5), 3.09 (m, 4H).
LCMS (ES +) m / z 355 (M + 1), R _t = 8.30, Method A
Example B2 Preparation of Compound 2

Lithium aluminum hydride (391 mg, 0.010 mol) was added portionwise to a solution of intermediate 6 (1.1 g, 0.0026 mol) in 55 ml THF at room temperature under argon. The mixture was stirred at room temperature for 18 hours. The reaction was quenched with MeOH at 0 ° C. Ice and diluted Rochelle salt solution were added and the mixture was extracted three times with DCM. The organic layer was separated, washed with saline, dried (MgSO ₄ ), filtered and the solvent was evaporated. The residue was purified by column chromatography over silica gel (40-63 μm) (eluent: DCM / MeOH / NH ₄ OH 97/3 / 0.5). The pure fractions were collected and the solvent was evaporated, yielding 121 mg (12%) of compound 2 as an orange foam.
¹ H NMR (300 MHz, CDCl ₃ ) δ 8.70 (d, 1H, J = 5.1), 8.18 (brs, 1H), 8.07 (d, 1H, J = 8.7), 8. 03 (d, 1H, J = 8.7), 7.66 (m, 2H), 7.41 (m, 2H), 7.22 (t, 1H, J = 7.7), 7.12 ( m, 3H), 6.63 (s, 1H), 6.56 (d, 1H, J = 8.4), 6.34 (dd, 1H, J = 8.4, J = 2.1), 4.06 (t, 2H, J = 7.8), 3.46 (t, 2H, J = 6.8), 3.12 (m, 4H).
MS (ES +) m / z 405 (M + 1).
Example B3 Preparation of compound 3

Lithium aluminum hydride (14 mg, 0.00036 mol) was added to a solution of intermediate 7 (74 mg, 0.00036 mol) in 1 ml THF at 0 ° C. under argon. The mixture was stirred at 0 ° C. for 1 h, quenched with a 5% solution of potassium hydrogen sulfate, and extracted twice with EtOAc. The organic layer was separated, washed with brine, dried (MgSO _4), filtered, and the solvent was evaporated, yielding 3-yl-acetaldehyde as an orange oil.

A mixture of intermediate 9 (200 mg, 0.00074 mol) and sodium cyanoborohydride (64 mg, 0.0010 mol) in 2.3 ml MeOH and 2 drops of acetic acid was added to the aldehyde (236 mg, 0.0015 mol). ) In 2 ml of MeOH. The reaction mixture was stirred at room temperature for 16 hours. The reaction was quenched with water, made alkaline with a saturated solution of sodium bicarbonate and extracted three times with EtOAc. The organic layer was separated, washed with saline, dried (MgSO ₄ ), filtered and the solvent was evaporated. The residue was purified by column chromatography over silica gel (40-63 μm) (eluent: EtOAc / MeOH 100/0 to 90/10). The pure fractions were collected and the solvent was evaporated, yielding 190 mg (44%) of compound 3 as an orange foam.
¹ H NMR (300 MHz, CDCl ₃ ) δ 8.42 (d, 1H, J = 5.7), 8.08 (brs, 1H), 7.79 (d, 1H, J = 2.4), 7. 63 (d, 1H, J = 7.8), 7.40 (d, 1H, J = 8.1), 7.25-7.07 (m, 5H), 6.59 (s, 1H), 6.48 (dd, 1H, J = 8.7, J = 2.1), 4.00 (m, 5H), 3.48 (t, 2H, J = 6.8), 3.12 (m , 4H).
MS (ES +) m / z 413 (M + 1).
Example B4 Preparation of compound 4

Sodium borohydride (92 mg, 0.0024 mol) was slowly added to a solution of compound 3 (100 mg, 0.00024 mol) in 3 ml MeOH. The mixture was stirred at room temperature for 1 hour, then at 80 ° C. for 4 hours, and again at room temperature for 85 hours. The reaction was quenched with water and the mixture was extracted with EtOAc. The organic layer was separated, washed with saline, dried (MgSO ₄ ), filtered and the solvent was evaporated. The residue was purified by column chromatography over silica gel (40-63 μm) (eluent: DCM / MeOH 90 / 10-85 / 15). The pure fractions were collected and the solvent was evaporated, yielding 50 mg (54%) of compound 4 as a yellow foam.
¹ H NMR (300 MHz, DMSO-d ₆ ) δ 10.83 (brs, 1H), 8.14 (d, 1H, J = 5.7), 7.54 (d, 1H, J = 7.8), 7.35 (d, 1H, J = 7.8), 7.22 (m, 2H), 7.14 (d, 1H, J = 2.1), 7.70 (dt, 1H, J = 7) .6, J = 1.2), 6.98 (dt, 1H, J = 7.5, J = 0.9), 6.86 (dd, 1H, J = 6.0, J = 2.4). ), 6.62 (d, 1H, J = 2.1), 6.45 (dd, 1H, J = 8.7, J = 2.2), 5.39 (brs, 2H), 4.49. (D, 2H, J = 4.0), 3.92 (t, 2H, J = 8.2), 3.30 (m, 2H), 3.06 (t, 2H, J = 8.1) , 2.96 (t, 2H, J = 7.5).
MS (ES +) m / z 385 (M + 1).
Example B5 Preparation of compound 5

Lithium aluminum hydride (14 mg, 0.00036 mol) was added to a solution of intermediate 7 (74 mg, 0.00036 mol) in 1 ml THF at 0 ° C. under argon. The mixture was stirred at 0 ° C. for 1 h, quenched with a 5% solution of potassium hydrogen sulfate, and extracted twice with EtOAc. The organic layer was separated, washed with brine, dried (MgSO _4), filtered, and the solvent was evaporated, yielding 3-yl-acetaldehyde as an orange oil.

A mixture of intermediate 13 (100 mg, 0.00037 mol) and sodium cyanoborohydride (33 mg, 0.00052 mol) in 1.5 ml MeOH and 2 drops acetic acid was added to the aldehyde (57 mg, 0.00036 mol). ) Solution (in 0.5 ml MeOH) was added dropwise. The reaction mixture was stirred at room temperature for 18 hours. The reaction was quenched with a saturated solution of sodium bicarbonate and extracted twice with EtOAc. The organic layer was separated, washed with saline, dried (MgSO ₄ ), filtered and the solvent was evaporated. The residue was purified by column chromatography over silica gel (40-63 μm) (eluent: DCM / MeOH 100/0 to 90/10). The pure fractions were collected and the solvent was evaporated, yielding 79 mg (52%) of compound 5 as a yellow foam.
¹ H NMR (300 MHz, CDCl ₃ ) δ 8.37 (brs, 1H), 8.15 (d, 1H, J = 5.8), 7.63 (d, 1H, J = 7.8), 7. 37 (d, 1H, J = 7.1), 7.21 (dt, 1H, J = 7.6, J = 1.2), 7.12 (dt, 1H, J = 7.6, J = 1.1), 7.07 (m, 2H), 6.83 (d, 1H, J = 8.5), 6.56 (d, 1H, J = 2.0), 6.40 (dd, 1H, J = 8.5, J = 2.3), 5.22 (t, 1H, J = 6.7), 4.19 (brs, 1H), 4.04 (m, 3H), 3. 44 (t, 2H, J = 6.7), 3.06 (m, 5H), 2.83 (m, 1H), 2.54 (m, 1H), 2.08 (m, 1H).
MS (ES +) m / z 411 (M + 1).
Example B6 Preparation of compound 6

It was added portionwise to a solution of intermediate 16 Lithium aluminum tetra hydride and (0.001 mol) under _{N 2} flow (0.002 mol) in THF (10ml). The mixture was stirred at reflux for 24 hours, poured into ice water and filtered over celite. Celite was washed with EtOAc. The filtrate was extracted with EtOAc. The organic layer was separated, dried (MgSO ₄ ), filtered and the solvent was evaporated. The residue (0.45 g) was purified by column chromatography over silica gel (10 μm) (eluent: DCM / MeOH / NH ₄ OH 95/5 / 0.5). The pure fractions were collected and the solvent was evaporated, yielding 0.042 g of compound 6.
¹ H NMR (DMSO-d6) δ 1.28 (6H, s), 2.95 (2H, t, J = 7.7 Hz), 3.27-3.34 (2H, m), 3.65 (2H , S), 5.38 (1H, br, t, J = 6.4 Hz), 6.45 (1H, dd, J = 4 Hz, 7.7 Hz), 6.57 (1H, d, J = 4 Hz) , 6.96-7.02 (3H, m), 7.07 (1H, t, J = 7.7 Hz), 7.15-7.5 (3H, m), 7.35 (1H, d, J = 7.7 Hz), 7.55 (1H, d, J = 7.7 Hz), 8.22 (1H, d, J = 7.7 Hz), 10.83 (1H, br, s)
MS (ES +) m / z 383 (M + 1), R _t = 9.17, Method A
Table F-1 lists the compounds prepared in one of the above examples.

C. Pharmacological Example A The ability of a compound to preserve p53 in 2780 cells was measured with a p53 enzyme immunoassay. The p53 assay is a “sandwich” enzyme immunoassay using two polyclonal antibodies. A polyclonal antibody specific for the p53 protein was immobilized on the surface of a plastic well. Any p53 present in the sample to be measured will bind to the capture antibody. Biotinylated detection polyclonal antibody will also recognize the p53 protein and bind to any p53 carried by the capture antibody. The detection antibody is in turn bound by horseradish peroxidase-conjugated streptavidin. Horseradish peroxidase catalyzes the conversion of the chromogenic substrate o-phenylenediamine, and its strength is proportional to the amount of p53 protein bound to the plate. The colored reaction product is quantified using a spectrophotometer. Quantification is achieved by construction of a standard curve using known concentrations of purified recombinant HIS-labeled p53 protein (see Example C.1).

  The cellular activity of the compound of formula (I) was determined against U87MG tumor cells using a colorimetric measurement for cytotoxicity or viability (see Example C.2).

  U87MG cells are human glioblastoma cells with wild type p53. In this cell line, MDM2 closely regulates p53 expression.

Example C. 1. p53 ELISA
A2780 cells (ATCC) were cultured in RPMI 1640 supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine and gentamicin in a humidified incubator containing 5% CO ₂ at 37 ° C.

A2780 cells were seeded at 20.000 cells per well in a 96-well plate, cultured for 24 hours, and treated with compounds for 16 hours at 37 ° C. in a humidified incubator. After incubation, the cells were washed once with phosphate buffered saline and 30 μl of low salt RIPA buffer (20 mM Tris, pH 7.0, 0.5 mM EDTA, 1% Nonidet P40, 0 per well). 0.5% DOC, 0.05% SDS, 1 mM PMSF, 1 μg / ml aprotinin and 0.5 μ / ml leupeptin). Plates were placed on ice for 30 minutes to terminate lysis. p53 protein was detected in delysates by using the following sandwich ELISA.

High binding polystyrene EIA / RIA 96 well plate (Costar 9018) with 50 μl per well of capture antibody pAB1801 (Abcam ab28-100) at a concentration of 1 μg / ml in coating buffer (0.1 M NaHCO ₃ , pH 8.2). Coated. The antibody was allowed to attach overnight at 4 ° C. The coated plate is washed once with phosphate buffered saline (PBS) /0.05% Tween 20, and 300 μl blocking buffer (PBS, 1% bovine serum albumin (BSA)) is incubated for 2 hours at room temperature. Over the course of time. Dilutions of purified recombinant HIS-labeled p53 protein ranging from 3 to 200 ng / ml were prepared in blocking buffer and used as standards.

Plates were washed twice with PBS / 0.05% Tween 20 and blocking buffer or standards were added at 80 μl / well. 20 μl lysis buffer was added to the standard. Samples were added to other wells at 20 μl lysate / well. After overnight incubation at 4 ° C., the plates were washed twice with PBS / 0.05% Tween 20. An aliquot of 100 μl of the second polyclonal antibody p53 (FL-393) (Tebubio, sc-6243) at a concentration of 1 μg / ml in blocking buffer was added to each well and allowed to attach for 2 hours at room temperature. Plates were washed 3 times with PBS / 0.05% Tween20. 0.04 μg / ml detection antibody anti-rabbit HRP (sc-2004, Tebbio) in PBS / 1% BSA was added and incubated for 1 hour at room temperature. The plate was washed 3 times with PBS / 0.05% Tween 20 and 100 μl substrate buffer was added (substrate buffer was 25 ml OPD buffer: 35 mM citric acid, 66 mM Na ₂ HPO ₄ , pH 5.6, Sigma. Prepared immediately before use by adding 1 tablet of 10 mg o-phenylenediamine (OPD) and 125 μl of 3% H ₂ O ₂ ). After 5-10 minutes, the color reaction was stopped by adding 50 μl stop buffer (1M H ₂ SO ₄ ) per well. Absorption at two wavelengths of 490/655 nm was measured using a Biorad microplate reader and the results were then analyzed.

  For each experiment, controls (containing no drug) and blank incubations (containing no cells or drugs) were performed in parallel. Blank values were subtracted from all control and sample values. For each sample, the p53 value (in absorption units) was expressed as a percentage of the p53 value present in the control. Storage rates higher than 140% were defined as significant. Here, the effect of the test compound is expressed as the lowest amount that gives at least 140% of the value for p53 present in the control (LAD) (see Table F-2).

Example C. 2. Proliferation assay All compounds to be tested were dissolved in DMSO and further dilutions were prepared in culture medium. The final DMSO concentration never exceeded 0.1% (v / v) in the cell proliferation assay. The control contained U87MG cells and DMSO without compound and the blank contained DMSO but no cells.

U87MG cells were seeded in 96-well cell culture plates at 3000 cells / well / 100 μl. After 24 hours, the medium was changed and compounds and / or solvents were added to a final volume of 200 μl. After 4 days of incubation, the medium was replaced with 200 μl fresh medium and cell proliferation was measured using an MTT-based assay. Therefore, 25 μl of MTT solution (0.5% MTT research grade from Serva in phosphate buffered saline) was added to each well and the cells were further incubated at 37 ° C. for 2 hours. The medium was then carefully aspirated and the blue MTT-formazan product was dissolved by adding 25 μl 0.1 M glycine and 100 μl DMSO to each well. The plate was shaken for an additional 10 minutes on a microplate shaker and then the absorbance at 540 nm was read on a Biorad microplate reader.

During the experimental period, the result for each experimental condition is the average of 3 replicate wells. For initial screening purposes, compounds were tested at a single fixed concentration of 10 -5 ^M. For effective compounds, the experiment to establish a complete concentration-response curve was repeated. For each experiment, a control (containing no drug) and a blank incubation (containing no cells or drugs) were performed in parallel. Blank values were subtracted from all control and sample values. For each sample, the mean value of cell growth (in absorption units) was expressed as a percentage of the mean value of control cell growth. Where appropriate, IC ₅₀ -values (concentration of drug required to reduce cell growth to 50% of control) were determined by probit analysis of the gradual data (Finney, DJ, Probit
Analyzes, 2nd Ed. (Chapter 10, Graded Responses, Campus University Press, Cambridge 1962). Here, the effect of the test compound is expressed as pIC ₅₀ (IC ₅₀ minus the logarithm of the value) (see Table F-2).

  In some experiments, proliferation assays were applied and used for 384-well culture plates (see Table F-2).

Example D.1. Composition Example: Film Coated Tablet
Preparation of tablet cores 100 g of a compound of formula (I), 570 g of lactose and 200 g of starch are mixed well, then in a solution of 5 g of sodium dodecyl sulfate and 10 g of polyvinyl-pyrrolidone (about 200 ml in water) Wet. Sift the wet powder mixture, dry and re-sieve. Then 100 g microcrystalline cellulose and 15 g hydrogenated vegetable oil are added. The whole is mixed well and compressed to give 10.000 tablets each containing 10 mg of the compound of formula (I).

Coating To a solution of 10 g of methylcellulose (in 75 ml of denatured ethanol) is added a solution of 5 g of ethylcellulose (in 150 ml of dichloromethane). Then 75 ml dichloromethane and 2.5 ml 1,2,3-propanetriol are added. 10 g of polyethylene glycol is melted and dissolved in 75 ml of dichloromethane. The latter solution is added to the former, then 2.5 g magnesium octadecanoate, 5 g polyvinylpyrrolidone and 30 ml concentrated pigment suspension are added and the whole is homogenized. In a coating apparatus, the tablet core is coated with the mixture thus obtained.

Claims (13)

Formula (I)

A compound thereof, its N-oxide form, addition salt or stereochemically isomeric form,
Where
m is 0, And direct bond is intended to,
n is are two der,
p is Ri 1 der,
t is 0, the element directly bonds are intended to,

Is ^-CR 8 = C <, and where the dotted line Ri 1 binding der, wherein ^{R 8} is hydrogen,
R ¹ and R ² are each hydrogen ;
R ³ and R ⁴ are each hydrogen ;
R ⁵ is hydrogen,
Each R ⁶ and ^{R 7} are independently water Motomata is selected from _{C 1-6} alkyl,
Z is

Is a group selected from, wherein each of R ¹⁰ and ^{R 11} are independently hydrogen, arsenate Dorokishi, _{C 1-6} alkyloxycarbonyl and hydroxy _{C 1-6} Ru selected alkyl Le or al. Wherein m is 0, n is 2 , p is 1, t is 0, R ¹ and R ² are each independently hydrogen, and R ³ and R ⁴ are each independently Hydrogen, R ⁵ is hydrogen, R ⁶ and R ⁷ are each independently hydrogen, Z is a group selected from (a-2) or (a-4), or R ¹⁰ and hydrogen R ¹¹ are each independently selected from hydroxy or hydroxyalkyl C _1-6 alkyl, claim 1 Symbol placement compound. The compound is compound no. 1, compound no. 4 or compound no. The compound according to claim 1 or 2 , which is 5.

4. A compound according to claim 1, 2 or 3 for use as a medicament. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound as claimed in claims 1 to 3 as an active ingredient. A method of preparing a pharmaceutical composition as claimed in claim 5 , wherein the pharmaceutically acceptable carrier and the compound as claimed in claims 1-3 are intimately mixed. for pharmaceutical manufacturing for the treatment of disorders mediated by p53-MDM2 interaction, use according to claim 1, 2 or 3 compounds described. A pharmaceutical composition comprising an anticancer agent and the compound according to claim 1, 2, 3 or 4. The intermediates of formula (II), formula (III) (wherein W is, for example, suitable leaving group such as halo) is reacted with intermediates,

A process for preparing a compound as claimed in claim 1, characterized in that In the presence of lithium aluminum hydride in a suitable solvent, an intermediate of formula (IV), of formula (I), whose p is 1 and is referred to herein as a compound of formula (Ia) Converting to a compound,

A process for preparing a compound as claimed in claim 1, characterized in that Reacting an appropriate carboxaldehyde of formula (VI) with an intermediate of formula (V) in an appropriate solvent in the presence of an appropriate reagent;

A process for preparing a compound as claimed in claim 1, characterized in that A compound of formula (I) wherein an intermediate of formula (II) is reacted with a suitable carboxaldehyde of formula (VII), where t is 1 and is referred to herein as a compound of formula (Ib) Forming,

A process for preparing a compound as claimed in claim 1, characterized in that   Use of a compound according to claims 1 to 3 for the manufacture of a medicament for inhibiting tumor growth.